Interactions between carbonic anhydrase and its inhibitors revealed by gelelectrophoresis and circular dichroism

Structural properties, and especially the differential stability, of comple xes between carbonic anhydrase (CA) and three sulfonamide inhibitors, aceta zolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel ele ctrophoresis either across a urea or a steady-state transverse sodium dodec yl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea , whereas dorzolamide, with a bulky and hydrophobic structure, is most stab le against the effects of SDS. At pH 7.4, complexes with dorzolamide show m inimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand , CA displays an increase in mobility above 0.05% SDS, lower in the presenc e than in the absence of excess ligand. The finding of a distinct pattern f or the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density . Under saturating conditions and in the presence of SDS, the surface charg e of all complexes is much lower than for unliganded, denatured CA. Circula r dichroism (CD) spectra clearly indicate that the increase in secondary st ructure and the decrease in tertiary structure brought about in CA by the p resence of low concentrations of SDS are largely prevented by complexing wi th the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological act ivities and also in the potential development of novel antagonist molecules .